Most HIV-infected people ultimately succumb to chronic infection if not adherent to combination antiretroviral therapy (cART). Some, however, known as elite controllers (EC), demonstrate superior virus control maintaining virtually undetectable viral loads even in the absence of treatment. While it is well known that HIV- specific cytotoxic lymphocytes (CTL) are critical for viral control and that HLA haplotype is associated with viral progression status, the mechanisms underlying EC virus control remain unknown. Recently, we showed that HIV-specific CTL from EC form better quality immunological synapses (IS) with HIV-infected target cells, with more rapid biogenesis of perforin and more effective lytic granule loading. We also found that engagement of inhibitory receptor programmed death-1 (PD-1, a marker for CTL exhaustion) on HIV-specific CTL specifically induces phosphorylation of the adaptor protein Chicken Tumor Virus #10 Regulator of Kinase (Crk). We hypothesize that chronic HIV leads to functional impairment of the CTL IS via upregulation of inhibitory receptors like PD-1 and that the resulting downstream phosphorylation of Crk, in turn, prevents CTL activation by disrupting the structure and signaling of the CTL-target cell synapse. Increased inhibitory receptor expression is one of the earliest markers of CTL exhaustion in chronic HIV. EC patients express lower levels of PD-1 than do their chronic progressor (CP) counterparts. In Aim 1 we will compare synapse structure and function in exhausted (CP) vs. non-exhausted (EC) HIV-specific CTL. HIV-specific CTL will be stimulated by a reductionist lipid bilayer system or by HIV-infected target cells in the presence o absence of PD-1's primary ligand PD-L1. IS will be visualized using super-resolution stimulated emission depletion microscopy and total internal reflection fluorescence (TIRF) microscopy. There are a number of important questions about IS that can only be addressed through super-resolution imaging (e.g. distribution of F-actin and lytic granules at IS). The proposed high-resolution, single cell assays will allow us to determine the specific impact of inhibitory recepto signaling on IS structure and function and will yield critical insights into how these relate to vial control (EC vs. CP) and haplotype. We hypothesize that upregulation of inhibitory receptor in exhausted cells results in increased pCrk, blocking critical Crk-dependent activation signals like Vav-1. Aim 2 will dissect the dynamics of signal integration at the IS of exhausted HIV-specific CTL, relating Crk phosphorylation and localization of key regulators, as well as cytotoxicity and viral inhibition, to inhibitory receptor expression level, haplotype, and viral progression status. Signaling will be tracked at a single-cell, synapse level using fluorescently tagged signal molecules and super-resolution STED microscopy. While other groups are attempting to boost T cell responses by targeting individual inhibitory receptors (e.g. PD-1 and CTLA-4), the proposed experiments will be the first to address a downstream, central signaling hub. It is our long-term goal to use the knowledge to augment T cell responses in HIV and other chronic viral infections.